The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.
Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.
A. Hyperbranched Polyglycerol Modification (SS-HPG)
B. Whole Blood Collection and Separation of Red Blood Cells (RBCs)
C. HPG Grafting to RBCs
D. Characterization of HPG Modified RBCs
I. Complement mediated lysis
II. The camouflage of major and minor antigens using MTS cards
III. Aqueous two phase partition measurements
IV. Osmotic fragility measurements
V. Flow cytometry measurements – Protection of Rhesus-D (RhD) antigen
VI. Flow cytometry measurements – Expression of CD47
Camouflage of Rhesus D antigen and CD47 RBC surface protein were quantified by flow cytometry using fluorescent labelled monoclonal antibodies, and a representative result is given in Figure 1. In case of HPG-grafted RBCs (grey), the intensity of the signal decreased (peak shifted to left) compared to the control RBCs (red & green) indicating a reduction in binding to antibodies to cell surface which indicate the masking of surface proteins.
Figure 1. Evaluation of the camouflage of surface antigens and proteins using flow cytometry. A) Rhesus D (RhD) protection: Black shaded peak represents the negative control (non-modified RBCs treated with PE-labelled IgG1), the green peak represent the positive control (non-modified RBCs treated with anti-RhD fluorescent antibody), and the grey peak represents HPG modified RBCs treated with the same amount of anti-RhD fluorescent antibody. B) CD47: Black shaded peak represents the negative control (non-modified RBCs treated with PE-labelled IgG1), the red peak represent the positive control (non-modified RBCs treated with anti-CD47 fluorescent antibody), and the grey peak represents HPG modified RBCs treated with the same amount of anti-CD47 fluorescent antibody. Click here to view larger figure.
Universal donor RBCs have great potential in enhancing blood availability and safety for blood transfusion therapy. RBCs are also considered promising drug delivery vehicles due to their long circulation and inherent biocompatibility 14, 15. Experiments presented in this paper evaluate the in vitro characteristics of HPG modified RBCs. The in vitro properties and in vivo circulation of HPG modified RBCs have been investigated in our group recently 8, 11. The partition of RBCs in Dextran500K/PEG8K aqueous two phase system, supplemented with NaCl and sodium phosphate, depends on the erythrocyte surface charge and on surface glycoprotein composition 16. Depending on the extent of RBC glycocalyx modification with HPGs, modified RBCs tend to partition from the lower dextran to the upper PEG phase. The two phase system provides a facile evaluation of the extent of RBC surface modification. Lysis of RBCs in autologous serum is significant test to investigate, whether HPG modified RBCs triggers complement activation, since introducing new materials to cells and biological surfaces renders them foreign and subject to immune system mediated lysis and clearance 17. The osmotic fragility experiment, on the other hand, indicates whether mechanical properties and deformability of RBC membrane has been compromised as a result of HPG grafting. The deformability of RBCs is important for the physiological function of O2 delivery to different parts of the body. In this experiment modified RBCs are exposed to deformation stress by treating with different concentrations of NaCl, and quantifying percent of lysed cells.
Flow cytometry is used to evaluate the extent of surface antigen and surface protein protection (Figure 1) by reacting a particular antigen on the surface of RBC with its corresponding fluorescent- labelled antibody. The extent of Rhesus D antigen protection is evaluated using flow cytometry. The masking of antigens is evident from the decrease in antibody binding to the RBCs. Rhesus D antigen masking is also evaluated by MTS cards. MTS cards are widely used in hematology laboratories in hospitals for phenotyping of RBCs. For example, when A-group RBC is located in A-type MTS card, cells agglutinate as a result of reaction with the corresponding monoclonal antibody. However, B-group blood does not agglutinate and travels to the bottom of the mini gel. The concept of agglutination was used to evaluate the level of protection that HPG grafting provides to RBCs. HPG-grafted RBCs penetrates the mini gel column, depending on the level of surface antigen protection.
The authors have nothing to disclose.
This research was funded by the Canadian Blood Services (CBS) and the Canadian Institutes of Health Science (CIHR) Research Partnership Fund. The authors thank the LMB Macromolecule Hub at the UBC Centre for Blood research for the use of their research facilities. The infrastructure facility is supported by the Canada foundation for Innovation (CFI) and the Michael Smith Foundation for Health Research (MSFHR). R. Chapanian is a recipient of (CIHR/CBS) postdoctoral fellowships in Transfusion Science and a recipient of MSFHR research trainee post doctoral fellowship. J.N. Kizhakkedathu is a recipient of MSFHR Career Investigator Scholar Award.
Glycidol | Sigma Aldrich | (ON, Canada) | |
Trimethylolpropane | Fluka | (ON, Canada) | |
Potassium methylate | Sigma Aldrich | (ON, Canada) | |
Anhydrous pyridine | Sigma Aldrich | (ON, Canada) | |
4-Dimethylaminopyridine | Sigma Aldrich | (ON, Canada) | |
Succinic anhydride | Sigma Aldrich | (ON, Canada) | |
Acetone | Fisher Scientific | (ON, Canada) | |
Anhydrous dimethyl formamide | Sigma Aldrich | (ON, Canada) | |
N-Hydroxysuccinimide | Sigma Aldrich | (ON, Canada) | |
N,N’-Diisopropylcarbodiimide | Sigma Aldrich | (ON, Canada) | |
MTS cards | Micro Typing System (MTS) cards (FL, USA) | ||
Dextran 500 kDa | Pharmacia Fine Chemicals | (Sweden) | |
PEG 8 kDa | Sigma Aldrich | (ON, Canada) | |
FITC monoclonal anti-Rhesus D (RhD) | Quotient Biodiagnostics | (PA, USA) | |
PE monoclonal anti-CD47 | BD Biosciences | (NJ, USA) | |
Drabkin’s reagent | Sigma Aldrich | (ON, Canada) | |
Table. Chemicals and reagents used for the grafting of HPG polymers to RBC membrane and their analysis. |